Angiogenesis, the process of new blood vessel development and formation, plays an important role in numerous physiological events, both normal and pathological. Angiogenesis occurs in response to specific signals and involves a complex process characterized by infiltration of the basal lamina by vascular endothelial cells in response to angiogenic growth signal(s), migration of the endothelial cells toward the source of the signal(s), and subsequent proliferation and formation of the capillary tube. Blood flow through the newly formed capillary is initiated after the endothelial cells come into contact and connect with a preexisting capillary.
Angiogenesis is indispensable for embryonic development, organogenesis, tissue regeneration and repair, wound healing and female reproductive processes (Folkman, J. And Shing, Y., J. Biol. Chem. 267:109931-10934, 1992; Folkman, J., Nature Medicine 1: 27-31, 1995). Meanwhile, angiogenesis is also one of the necessary factors that cause the progression and deterioration of many pathological disorders including cancer growth and metastasis, cardiovascular disease, diabetic retinopathy, rheumatoid arthritis, etc. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis.
Angiogenesis is a complex multi-stage process that includes proliferation, migration and differentiation of endothelial cells, proteolytic degradation of the basement membrane, differentiation and migration of endothelial cells into the surrounding stroma, and finally formation of vasculature and new capillaries. The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate (Rastinejad et al., 1989, Cell 56:345-355).
Angiogenesis stimulators that can be mentioned include vascular endothelial growth factor (VEGF), vascular permeability factor (VPF), fibroblast growth factor (FGF-1 and -2), etc. On the other hand, some angiogenesis inhibitors have also been found and identified, which includes a 29 kDa fragment of fibronectin, thrombospondin (TSP-1), platelet factor 4, a 16 kDa fragment of prolactin, and a 38 kDa fragment of plasminogen and the like. In particular, O'Reilly et al. identified and characterized an internal 38 kDa fragment of plasminogen as angiostatin and a 20 kDa globular C-terminal of collagen XVIII as endostatin. It is suggested based on the current research results that the angiogenesis phenotype in the tissue depends on the dynamic equilibrium of angiogenesis stimulator and inhibitors in the local tissue environment (Folkman, J., N. Engl. J. Med. 333: 1757-1763, 1995).
Particularly interesting is that recent research shows that most angiogenesis inhibitors as mentioned above display the inhibitory activity of endothelial cell proliferation only after their parent proteins are hydrolyzed and form terminal or internal fragments. Thus, it is suggested that protein hydrolysis by endogenous peptidases plays a key role in the expression of their biological activities (O'Reilly, M. S. et al., Cell 88:277-285, 1997).
As a 20 kDa carboxyl terminal fragment of collagen XVIII, endostatin is a special inhibitor of endothelial cell proliferation and migration, and it also markedly inhibits the growth of many kinds of cancers (O'Reilly, M. S. et al., Cell 88: 277-285, 1997; U.S. Pat. No. 5,854,205). It was shown that repeated endostatin administration leads to prolonged stable state of mice cancers, and there was no induction of drug resistance (Boehm, T. et al., Nature 390:404-407, 1997). It was further shown that endostatin causes cells to be quiescent at cell cycle G1 phase and specifically induces apoptosis of endothelial cells (Dhanabalk, M. et al., Biochem. Biophys. Res. Commun. 258: 345-352, 1999).
Endostatin was initially isolated from a hemangioendothelioma cell line for its ability to inhibit the proliferation of capillary endothelial cells (O'Reilly, M. S. et al., Cell 88:277-285, 1997). Based on the analysis of its nucleotide sequence, O'Reilly et al. further expressed endostatin protein in an E. coli expression system in un-refolded form, and it is believed that the unfolded purified protein facilitates its prolonged release at the subcutaneous injection site. The authors also mentioned that when endostatin was refolded by a standard method and solublized into tissue culture media, it strongly inhibited the proliferation of endothelial cells. Unfortunately, about 99% of protein was lost during protein refolding. In addition, though it has been reported that protein having anti-angiogenesis activity can be expressed in prokaryotes, the product can hardly refold into soluble form and tends to precipitate out of the solution. Further, cloning and expressing soluble recombinant endostatin in a yeast (Pichia pastoris) system were also reported (see, for example, Dhanabal, M. Et al., Cancer Res. 59: 189-197, 1999).
It has been shown that the requisite effective amount of endostatin expressed in yeast system is astonishingly high, about 240-600 mg/m2/person in recent clinical trials. It is reasonable to speculate that such a high dose of drug would place a great and burdensome demand on large scale manufacturing of the drug for clinical trials and on industrial production in the future sales and marketing of the drug made from endostatin, even though it appears the high dose might be safe as determined in mouse and monkeys. There have been efforts made in reducing the dosage of endostatin and increasing the effect of endostatin by using continuous infusion with pumps in clinical trials. However, this approach can cause great discomfort and inconvenience for the patients and it does not lead to an obviously improved clinical result.
In U.S. patent application Ser. No. 10/313,638, applicants disclosed a novel modified endostatin produced in E. coli, and more importantly, the E. coli produced endostatin has better in vivo stability and biological activity than native endostatin produced in yeast, thereby markedly improving its pharmaceutical activity and substantially decreasing the requisite clinical administration doses. These findings can be seen from our previous experimental results, and pharmacological and pharmacokinetics studies. Other studies have shown that the administration of endostatin to tumor-bearing mice leads to significant tumor regression, and no toxicity or drug resistance has been observed even after multiple treatment cycles (Boehm et al., 1997, Nature 390(6658): 404-407). The fact that endostatin targets genetically stable endothelial cells and inhibits a variety of solid tumors makes it a very attractive candidate for anticancer therapy (Fidler and Ellis, 1994, Cell 79(2): 185-8; Gastl et al., 1997, Oncology 54(3): 177-84; van Hinsbergh et al., 1999, Ann Oncol 10 Suppl 4:60-3). In addition, angiogenesis inhibitors have been shown to be more effective when combined with radiation and chemotherapeutic agents (Klement, 2000, J. Clin Invest, 105(8) R15-24. Browder, 2000, Cancer Res. 6-(7) 1878-86, Arap et al., 1998, Science 279 (5349): 377-80; Mauceri et al., 1998, Nature 394 (6690): 287-91).
However, there have been no reports demonstrating the clinical efficacy of endostatin, especially E. coli produced modified endostatin. Currently, treatments such as surgery, chemotherapy, or radiotherapy have only achieved limited in treating cancer in human and animals. The present invention attempts to address the issue of inadequate and insufficient clinical efficacy of existing cancer therapy, and discloses a new method of therapy which takes advantage of the therapeutic use of the modified endostatin in treating angiogenesis-related diseases, in particular, cancer.